Identification of Human Leukocytic Antigens (HLA)
Accurate HLA typing is essential for matching donor and recipient in organ or marrow transplantation (4) to prevent the development of acute graft-versus-host disease (GVHD). This is generally accomplished by standard serological typing (2). Recent studies have demonstrated that DNA genotyping can provide more accurate and definitive result (7, 9, 8). Results of HLA-DQ, DR and DP genotyping provided data for accurate matching which is necessary in selecting potential organ donors (3). HLA genotyping using sequence-specific primers polymerase chain reaction (SSP-PCR) amplifications has been previously reported. However, due to highly polymorphic nature of HLA-DQ, DR and DP loci, the number of SSP required will be in the hundreds, and, therefore, exceed the limit of multiplex-PCR for efficient PCR amplification. To ensure the results of HLA genotyping have practical clinical application, multiple of 50 to 100 of separate PCR reactions have to be setup. A kit that is available on the market today includes an amplification process of carrying out a 96 PCR separate reactions, then followed by analysis on gel electrophoresis size separation. This is not only time consuming and costly but prone to error due to the complexity of the reaction setup and sizing uncertainty of the gel electrophoresis. Thus, DNA sequencing is still considered the method of choice for accurate genotyping of the HLA cluster. Unfortunately, because of the existence of highly homologous sequence of pseudogene(s) that may be co-amplified during the polymerase chain reaction (PCR) amplification process, accurate genotyping by DNA sequencing alone may prove more difficult and costly. U.S. Pat. Nos. 5,471,547 and 6,020,187, issued to J. W. O. Tam, disclose a fast annealing process that uses a very inexpensive device for accurate mutation detection, genotyping and fingerprinting analysis. The present invention discloses a method of rapidly analyzing HLA loci of DP, DR and DQ beta sequences by ASO oligo-probes using an improved flow-through format.
DNA Fingerprinting for Rapid Identification of Human Beings and Organisms
DNA fingerprinting by restriction fragment length polymorphism (RFLP) was first introduced in 1985 (12) for human identification, and was subsequently applied to identification of other organisms. In practical applications, DNA fingerprinting has been widely accepted as the best forensic tool for identification of suspects in criminal cases, for paternity disputes and for establishing or verifying the identity of a person. The time consuming RFLP method has been gradually replaced by high throughput automated processes. Using PCR amplification for analyzing the number of short tandem repeat (STR), first discovered in 1991 (11), from 10, 16, 18 or more loci in the human genome, single cell identification is now possible. However, both STR and/or variable number of tandem repeats (VNTR) are relatively expensive because these methods require the use of sophisticated equipment, and labor intensive and time consuming process like the Southern blotting hybridization. Sporadic mutations (10) may reduce the accuracy and the power for definitive identification. Furthermore, STR data suggested that the frequency of mutation, particularly in cancer patients, is not uncommon. Hence, new alternative method is needed. Single nucleotide polymorphism (SNP) genotyping provides greater discriminating power by selecting an appropriate number of SNPs at unlink loci, and the mutation frequency in each locus (site) is lower than VNTR or STR systems for forensic or individual personal identification. This invention presents a method of making rapid, definitive biometric identification of an individual, such as a human being, animal, plant or any organism, using SNP genotyping.
HPV Genotyping
Human papillomaviruses (HPV) attack mucosal cells and is very heterogeneous; about 200 different genotypes have been reported in world-wide population with different prevalence among the species. According to their disease causing severity HPV strains are categorized into high risk (HR) and low risk (LR) strains. HR types are more frequently found in premalignant or malignant cervical lesions, while LR types in benign cases (4). There are about half million new cases of cervical cancer and estimated deaths of over 250,000 annually. Thus, HPV infection and cervical cancer remain one of the major female cancers worldwide (5). For this reason, HPV screening for the cervical cancer prevention is recommended since it is more sensitive than cytology (6,7).
Clinical studies revealed that 99.7% of cervical carcinoma was found to be associated with HR-HPV infections. Although HPV may be cleared for most infections, persistence of HR-HPV infection would lead to the development of high grade cervical intraepithelial neoplasia (i.e. CIN I; CIN II or CIN III) and the progression to cervical cancer (7, 8). Hence assays targeting to HPV viral DNA are developed and commercially available. The U.S. FDA has approved Hybrid Capture 2 (HC2) system (Digene Corporation, Gaithersburg, Md., USA), and the AMPLICOR HPV Test (Roche Molecular Diagnostics, Branchburg, N.J., USA). These screening tests identify the presence of either HR or LR of the most prevalent types of HPV in groups without differentiating genotypes. Although these tests provide good assays for HPV screening, the inability of genotyping HPV limits their use for clinical diagnostic and prognostic applications because the disease causing severity is very different, among them type 16 is most serve followed by 18, 33, 45 and 59 and so on (8). Furthermore persistent infection with the same HPV genotype will further increase the risk for cervical cancer (9). Hence effective and affordable genotyping assays are most needed.
The LINEAR ARRAY HPV Genotyping Test (covering 37 types by Roche Molecular Diagnostics, Branchburg, N.J., USA), and the INNO-LiPA HPV Genotyping covering 28 types (Innogenetics, Belgium), could complement either HC2 or the AMPLICOR HPV Test assay in distinguishing specific genotypes, and identify infections involving multiple genotypes. Given the facts that 1) oncogenic potential varies among HR-HPVs; 2) risk is elevated with persistent infection of the same genotypes; and 3) HPV vaccines available could not prevent all types of HPV infections, HPV genotyping is needed in addition to just generic screening for the presence or absence of HPV viruses. However, the above assays are expensive and still use conventional hybridization processes that require high running cost and hands on time. Most importantly, these genotyping kits lack the ability to detect HPV viruses outside their predetermined genotyping panel and therefore results in higher false negative rate in HPV screening. Hence, there is a need for HPV genotyping assays that are faster, cheaper, cover more genotypes and therefore more effective as an affordable alternative for HPV detection.
Tubuculosis Detection
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB). The emergence of anti-tuberculosis drug resistance is a serious problem for TB control programs in industrialized and developing countries alike. In addition to its clinical and epidemiological importance, multidrug-resistant tuberculosis (DR-MTB) has important economic impact because the cost of treatment is higher than that of normal MTB. DR-MTB is defined as resistance to at least two of the most effective first-line drugs, rifampin (RIF) and isoniazid (INH). These cases are on the rise globally, resulting in significant morbidity and mortality. Resistance to first-line anti-tuberculosis drugs has been linked to mutations in several genes in the MTB genome: rpoB for RIF resistance; katG and inhA for INH resistance.
Several technologies exist for DR-MTB detection. Conventional phenotypic Drug susceptibility testing (DST) remains the gold standard for MTB drug resistance testing but can take up to eight weeks to complete. Treatment would often begin before the results are confirmed. Conventional DNA sequencing for detection of MTB drug resistance mutations is not routinely available in the commercial setting because of the expense, necessary expertise and time-consuming nature. Alternatively, by utilizing polymerase chain reaction (PCR) and “Flow-through” hybridization technology, the present invention provides GenoFlow™ DR-MTB Array Test that can detect MTB drug resistance (from DNA extraction to analysis) within 4 hours with highly specific and sensitive result.
Beta Thalassemia Detection
Beta Thalassemia is a common hemoglobinopathy that is caused by autosomal mutations, which can be sub-divided into categories depending on the extent to which beta-globin production is affected. Some mutations result in mild reductions in the production of beta-globin (denoted as β+), whereas some cause absolute silencing of the beta-globin gene, or the production of non-functional beta-globin (denoted as β0). It is estimated that around 1.5% of the world's population are beta-thalassemia heterozygotes, or carriers, and the coupling of two carriers may produce offsprings that are homozygous for the disease. Disease severity manifests in three different degrees: from mild (thalassemia minor, β+/β or β0/β) to intermediary (thalassemia intermedia, β+/β or β0/β to severe (thalassemia major, β+/β+, β0/β+ or β0/β0). Individuals that inherit the thalassemia major trait develop hypochromic anemia, and may require life-long blood transfusion. The best method of beta-thalassemia control is prevention of birth of thalassemic children, by genetic counseling of prospective parents and prenatal diagnosis, which have proven to be the most effective management for the disease.
Beta-thalassemia is endemic in temperate regions such as Mediterranean countries, Middle East and Southeast Asia. The molecular bases for beta-thalassemia has been extensively studied and micro-mapped to point mutations along the beta-globin gene. Different geographic regions have different mutation spectra, and the frequency of mutations is not homogeneous in each region.
Current detection technologies include microarray, AS-PCR and direct sequencing. Alternatively, DNA testing using PCR with reverse dot blot hybridization has offered a highly sensitive and specific method for beta-globin mutant detection and has further permitted for the genotyping which assist the diagnosis of beta-thalassemia.
Hepatitis B Virus Detection
HBV is the smallest DNA virus comprising 3000 nucleotides surrounded by a protein capsid. It can be transmitted through contact with blood or body fluids of an infected individual. Approximately 2 billion people are infected with HBV; in which over 350 million become HBV carriers. About one-fifth would develop HBV-related cirrhosis or eventually liver cancer. Detection of HBV can be diagnosed by surface antigen using simple blood tests and HBV DNA measurement using polymerase chain reaction (PCR).
In addition, analysis of the divergence of HBV genome sequences has led to the identification of 10 HBV genotypes (A to J) and several subtypes. HBV genotypes and subtypes show a distinct geographical distribution. It has been discovered that pathogenic and therapeutic differences exist among various HBV genotypes. The determination of HBV genotypes can provide information for the management of chronic HBV infection and pre-treatment evaluation. DNA sequencing has been considered as the gold standard method for the detection of HBV genotypes. However, it is less efficient in detecting mixed genotypes. Alternatively, using molecular DNA techniques has offered a highly sensitive and specific method for HBV genotype detection.
Detection of Sexually Transmitted Disease
Sexually transmitted diseases (STDs) are common world-wide. According to 2005 WHO estimates, 448 million new cases of curable STDs (syphilis, gonorrhoea, chlamydia and trichomoniasis) occur annually throughout the world in adults aged 15-49 years. In Hong Kong, around 1 in 550 was diagnosed with Sexually Transmitted diseases (not including HIV) in 2010. For pregnant woman, STDs can infect her baby before, during, or after the baby's birth. Untreated gonorrhea and chlamydia may result in infertility. Therefore, pre-married couples and pre-pregnancy women are also potential users of STDs detection. In addition, China is also a potential market of STDs diagnosis, as the number of STDs patient increase dramatically in past 20 years. The fast, low-price and accurate STDs diagnostic kit is necessary for controlling STDs in China.
Thrombophilia Identification
Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis. Studies report that about 40% of thrombophilia cases presenting with thrombosis are inherited and has been shown to be a risk factor for cardiovascular disease including venous thrombosis as well as reproductive disorders including recurrent miscarriage. There is a growing view that inherited thrombophilia would predispose women towards adverse pregnancy outcomes including recurrent pregnancy losses, intrauterine fetal death, intrauterine growth retardation, preeclampsia and placental abruption.
Over the past two decades, several gene variants have been identified for inherited thrombophilia. The top 4 mutations are located at Factor V Leiden (FVL) [1691G>A], Factor II (Prothrombin) [20210G>A], Methylenetetrahydrofolate Reductase (MTHFR) [677C>T] and Methylenetetrahydrofolate Reductase (MTHFR) [1298A>C].
Testing for these genetic variations can greatly contribute to identification of high risk population of venous thrombosis and recurrent miscarriage, so as to help lowering the individual's risk and preventing the diseases and also providing prophylactic treatment guidance.